As the Covid-19 disease emerged, clinical observations have shown that the pathogenesis was frequently associated with coagulopathy and thrombotic complications. Initial studies have reported patients meeting the International Society of Hemostasis and Thrombosis (ISTH) criteria for DIC . However, soon it was shown that there were remarkable differences between Covid associated coagulopathy (CAC) and traditional sepsis-associated DIC. In contrast to DIC, the fibrin degradation products such as D-dimer are increased accompanied by a modest decrease in platelets and slight or no prolongation in PT and aPTT [2, 18, 19]. Another important key factor that is involved in CAC pathophysiology is the endothelium. We know that normal endothelial function refers to the control of the vascular tonus, permeability, cell adhesion, and anticoagulation . Upon stimulation and attachment to ACE2 by SARS-CoV2; the endothelium turns into a state called ‘endothelial dysfunction’ that represents a proinflammatory and prothrombotic state . We had postulated that Covid-19 pathogenesis was not only caused by respiratory epithelial but also endothelial involvement at the lungs and published the pathogenesis of pulmonary intravascular coagulation-PIC . The autopsy series had also supported that endothelium is heavily causing small and firm microthrombi in sections of peripheral lung parenchyma even in the absence of gross inflammation [6, 7, 8, 9].
In our study, we postulated that the CAC starts with endothelial dysfunction and is accompanied by increased fibrinolysis followed by fibrinolytic shut-down and loss of natural anticoagulants making the clinical picture more serious. We analyzed the hemostatic parameters at admission. Fibrinogen levels (presumably due to increase as an acute phase reactant) and D-dimer levels were found to be increased in the patient group in accordance with the previous studies [22, 23]. Prothrombin time was slightly prolonged as well. When we evaluated the endothelial dysfunction; we found that EPCR, sE-selectin, sP-selectin, PAI-1, TPA were significantly increased in Covid-19 patients when compared to normal controls. These biomarkers also showed an increasing trend between non-ICU versus ICU and survivor versus non-survivor groups, however, only a statistically significant difference was seen in median TPA levels between ICU vs non-ICU groups (p < 0.001). These findings suggested that early endotheliopathy at admission plays an important role in CAC.
The Fibrinolytic system is controlled by plasminogen activators and inhibitors with a result of plasminogen converted to plasmin. Tissue plasminogen activator, urea plasminogen activator (uPA), and their inhibitor PAI-1 are the key players of this system. Plasma TPA and PAI-1 levels were shown to be increased in patients with Covid-19 and worse outcomes . In our cohort, despite the levels of the mentioned biomarkers were different among ICU, non-ICU, and control groups, the only statistically different marker was TPA between ICU and non-ICU groups. When we compared survivor and non-survivor groups, median levels of none of the biomarkers including TPA were found to be different among groups. This finding may be caused by the small number of patients in the non-survivor group. The major source of TPA in Covid-19 patients is likely to be the endothelium, PAI-1 is secreted from the endothelium and to a lesser extent from the platelets that make us think the endothelial activation-dysfunction is accompanied by platelet activation at early stages. Neutrophil extracellular traps present at inflammatory infiltration of lungs may also contribute to the secretion of PAI-1 as can be speculated from a study by Zuo et al . They have shown a correlation between absolute neutrophil counts and TPA/PAI-1 levels. Another fibrinolytic system element that we analyzed was soluble fibrin monomer complex (SFMC) that is increased in hypercoagulable states and at the early stages of DIC [25, 26]. It was also shown to be a useful marker in hypercoagulable states with increased fibrinolysis . In our cohort, median SFMC levels were significantly lower in Covid-19 patients, especially patients in the ICU group, when comparing to controls showing the fibrinolytic shut-down is present even at admission.
Platelet activation and danger signal HMGBP-1:
When we analyzed the platelet activation markers, we evaluated the levels of sP-selectin (which is an activation marker of both endothelium and platelets), PDMP, and β-thromboglobulin. Soluble P-selectin was increased in Covid-19 patients especially in the ICU group when compared to controls in our cohort (p < 0.001). The molecular interaction between sP-selectin on platelets and endothelial cells is known to rapidly cause the expression of tissue factor (TF) on monocytes. The increase in sP-selectin may show an early activation of platelets causing localized intravascular micro-thrombosis. Our findings were consistent with the study by Agrati et al who had also shown increased levels of sP-selectin in Covid-19 patients . These findings make us think that in Covid-19 pathogenesis, the endothelium is activated initially causing an interaction between endothelium and platelets leading to further activation of the inflammatory system and resulting in fibrin formation.
An interesting finding of our study was that the median PDMP levels were found to be lower in Covid-19 patients when compared to the control group. Circulating microparticles are increased in malignant and non-malignant Covid-19 patients recently . Another study by Rausch et al showed that peripheral blood mononuclear cell binding to microparticles correlated with disease severity in Covid-19 patients . Our results were in contrast with the previous literature as we had expected an increase in PDMPs in Covid-19 patients. It was recently shown that platelets can form aggregates with activated leukocytes and monocytes in Covid-19 patients . The decrease in PDMP levels may be explained by the corporation of PDMPs to these aggregates, therefore, causing a decrease in plasma levels, however, this hypothesis needs to be tested with further studies.
The other biomarker that we did not find any difference among patient and control groups was β-thromboglobulin. High molecular weight group box protein-1 (HMGBP-1) and β-thromboglobulin were not studied previously in Covid-19 patients, our findings showed no difference of both markers in patient and control groups.
Natural anticoagulant proteins:
We studied soluble thrombomodulin (both as an endothelial marker and an anticoagulant protein), AT, and protein C as natural anticoagulants. Despite the median levels of s-thrombomudulin was not different between ICU, non-ICU, and control groups, in a study by Goshua et al, it was shown to be correlated with the hospital discharge status and mortality in Covid-19 patients . One of our study limitations is that we could not evaluate the correlation of levels of biomarkers with mortality as our cohort only contained 6 patients in the non-survivor group. Median AT and protein C levels were found to be significantly decreased in patient group compared to the control group and this decrease was more profound in the ICU group showing an initial decrease of the anticoagulant system.
Limitations of our study:
Our study had several limitations. The number of patients in the cohort was small, even smaller in subgroups. The budget of our grant limited the number of patients studied, therefore we could only include the minimum number of patient and control subjects that can enable statistical analysis. Despite we tried to evaluate different parts of coagulation activation, we could not correlate these biomarkers with a viscoelastic evaluation such as TEG or ROTEM as we lacked this system. For PDMP levels it would have been more specific to evaluate them via flow cytometry, however, this method required immediate evaluation of cells and microparticles with a fresh sample, we could only collect and freeze samples and study all in once therefore we preferred the ELISA method in every marker to have the opportunity to study them together. Another limitation was that the number of patients in ICU, non-ICU, survivor, and non-survivor subgroups were low, disabling us to evaluate the effect of the markers over survival and ICU stay. Because of a limited budget of the grant, we could not evaluate tissue factor, von Willebrand factor, and other coagulation factor levels. The evaluation of all factors will also have a reflection on how coagulation is activated in Covid-19.